The consumption of tobacco products leads to the elevation of several health risks. Smokers have a higher likelihood of developing cardiovascular disease, respiratory disease, and several types of cancer. As a result, approximately 20% of all deaths in the United States are associated with tobacco products. Despite the clear hazards, it is estimated that more than 40 million adults in the United States are smokers. One of the primary barriers to decreasing the number of smokers is that nicotine, a component of tobacco, is highly addictive. We propose a novel approach to elucidate the mechanism of nicotine addiction and to identify target nAChR assemblies and phenotypes for nicotine cessation therapeutics. In the R21 phase, we will focus on the development of a novel approach to monitor the assembly and trafficking of nicotinic receptors at the single molecule level. Nicotinic receptors cannot be purified outside of the cellular environment making them inaccessible to common single molecule techniques that require the purification and reconstitution of proteins. We will utilize receptors isolated in cell derived vesicles. Isolation of vesicles from different organelles will allow us to determine nicotine and therapeutic ligands affect the assembly and trafficking of nAChRs. Identification of the specific structural assemblies targeted by existing therapeutics will allow us to identify specific targets for cessation therapeutics. In the R33 phase, we will extend studies to receptors generated in live animals by isolating vesicles from different regions of the brain of nicotinic receptor GFP knock-mice. These will be the first single molecule studies of nicotinic receptors generated in live animals. We will be able to correlate the assembly of different receptor subtypes to specific brain regions. These studies will for the first time allow us to perform single molecule fluorescence studies on brain derived nAChRs. Understanding how existing therapeutics alter structural assembly in specific brain regions will provide an understanding of which nAChRs to target and the structural assembly they should induce. This will provide insight into the mechanism of nicotine addiction and on the receptor phenotypes optimally induced by cessation therapeutics.